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The Functions of SETD5 and Mir-221 in Embryonic Stem Cell Differentiation

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The Functions of SETD5 and Mir-221 in Embryonic Stem Cell Differentiation The Texas Medical Center Library DigitalCommons@TMC The University of Texas MD Anderson Cancer Center UTHealth Graduate School of The University of Texas MD Anderson Cancer Biomedical Sciences Dissertations and Theses Center UTHealth Graduate School of (Open Access) Biomedical Sciences 12-2017 The functions of SETD5 and miR-221 in embryonic stem cell differentiation Tsai-Yu Chen Follow this and additional works at: https://digitalcommons.library.tmc.edu/utgsbs_dissertations Part of the Medicine and Health Sciences Commons, Molecular Biology Commons, and the Other Cell and Developmental Biology Commons Recommended Citation Chen, Tsai-Yu, "The functions of SETD5 and miR-221 in embryonic stem cell differentiation" (2017). The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access). 817. https://digitalcommons.library.tmc.edu/utgsbs_dissertations/817 This Dissertation (PhD) is brought to you for free and open access by the The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has been accepted for inclusion in The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access) by an authorized administrator of DigitalCommons@TMC. For more information, please contact [email protected]. THE FUNCTIONS OF SETD5 AND MIR-221 IN EMBRYONIC STEM CELL DIFFERENTIATION Tsai-Yu Chen, M.S. APPROVED: Min Gyu Lee, Ph.D. Advisory Professor Xiaobing Shi, Ph.D. Jae-Il Park, Ph.D. Min Sup Song, Ph.D. Liuqing Yang, Ph.D. APPROVED: Dean, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences I TITLE PAGE THE FUNCTIONS OF SETD5 AND MIR-221 IN EMBRYONIC STEM CELL DIFFERENTIATION A DISSERTATION Presented to the Faculty of The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY by Tsai-Yu Chen, M.S. Houston, Texas December, 2017 II ACKNOWLEDGEMENTS ACKNOWLEDGEMENT I would like to first thank my mentor, Dr. Min Gyu Lee, for giving me the opportunity to pursue my degree in his laboratory. He brought me into the epigenetic field and really opened my eyes as a scientist. I always remembered the most important thing he told me, “Control! Control! Control! Always include controls in the experiment.” By following his guidance, I learned numerous new things, not only in the scientific field but also precious life lessons. I would like to express my sincere gratitude to all the faculty members that have served on my committees, Dr. Hui-Kuan Lin, Dr. Jessica Tyler, Dr. Xiaobing Shi, Dr. Jae-Il Park, Dr. Min Sup Song and Dr. Liuqing Yang. Your suggestions and guidance really provided me great directions and inspirations to finish my projects and my presentation skills. I would like to thank all the former and current members in Dr. Lee’s laboratory. Dr. Na Li, Dr. Hunain Alam, Dr. Jaehwan Kim, Dr. Bingnan Gu, Dr. Eunah Kim and Sarah Wu. Thanks for everything during the past 7 years in the lab. It has been a privilege to work with you everyday in the lab. I’ve learned a lot from every one of you. Moreover, I want to especially thank Dr. Sunghun Lee and Dr. Shilpa Dhar. I’ve learned everything about my projects from you and you two provided me not only your knowledge but also the great patience to help me during the past several years. I could not finish my PhD without your help. I would like to thank my parents, Mei-Yin Lee and Chu-I Chen for your unconditional love and support to allow me to pursue my dream in the USA. Last but not least, I would like to thank my lovely wife Tsun-Hsuan Chen. I am the luckiest person in the world to meet you and marry you. You bring me happiness every day and accompany me through all the difficulties. Thank you and I love you. III ABSTRACT THE FUNCTION OF SETD5 AND MIR-221 IN EMBRYONIC STEM CELL DIFFERENTIATION Tsai-Yu Chen, M.S. Advisory Professor: Min Gyu Lee, Ph.D. Embryonic stem cells (ESCs) are a widely used model system to study cellular differentiation because of their pluripotent characteristics, and ESC differentiation is an epigenetic process. In an effort to identify a new epigenetic factor that is required for ESC differentiation, the function of SETD5 in ESCs was studied for this thesis. Results show that SETD5 is essential for retinoic acid (RA)-induced differentiation of mouse ESCs and for RA-induced expression of critical developmental genes (e.g., Hoxa1 and Hoxa2) and neuron-related genes (e.g., Nestin and Pax6). SETD5 was upregulated during ESC differentiation. Additional results demonstrated that SETD5 bound to RAR-α upon RA treatment and was recruited to a retinoic acid response element (RARE) for Hoxa1 and Hoxa2 activation. Methyltransferase assay using recombinant SETD5 and SETD5 complex showed that SETD5 was catalytically inactive, although it has a putative catalytic domain called SET. The transcription coactivator HCF1 was identified as a major SETD5-interacting protein. Depletion of HCF1 inhibited RA-induced ESC differentiation and RA-induced expression of SETD5-regulated genes. Chromatin immunoprecipitation assay provided evidence that HCF1 was localized to the SETD5-bound RARE region after RA treatment. These findings reveal a previously unknown ESC differentiation mechanism in which SETD5 facilitates mouse ESC differentiation by activating differentiation-specific genes via cooperation with HCF1. IV In a related but independent study, we show that miR-221-3p and miR-221-5p, which are encoded by the miR-221 gene, are new anti-stemness miRNAs whose expression levels in mouse ESCs are directly repressed by the epigenetic modifier and pluripotent factor PRMT7. Notably, both miR-221-3p and miR-221-5p can target the 3’ untranslated regions of the major pluripotent factors Oct4, Nanog and Sox2 to antagonize mouse ESC stemness while miR-221-5p additionally silences expression of its transcriptional repressor PRMT7. Transfection of miR-221-3p and miR- 221-5p mimics induced spontaneous differentiation of mouse ESCs. CRISPR-mediated miR-221 deletion and anti-sense miR-221 inhibitors inhibited spontaneous differentiation of PRMT7- depleted mouse ESCs. These results reveal that PRMT7-mediated repression of miR-221-3p and miR-221-5p is critical for maintaining mouse ESC stemness. Taken together, these two studies establish SETD5 and miR-221 as novel anti-stemness regulators that play a pro-differentiation role in ESC differentiation. V TABLE OF CONTENTS Title Page ...................................................................................................................................... II Acknowledgements ...................................................................................................................... III Abstract ........................................................................................................................................ IV Table of Contents ......................................................................................................................... VI List of Figures ............................................................................................................................... X List of Tables .............................................................................................................................. XII CHAPTER 1 Introduction.............................................................................................................. 1 1.1 General Background of embryonic stem cells ..................................................................... 2 1.2 General Background of Epigenetics ..................................................................................... 3 1.3 Post-translational Modifications of Histones ....................................................................... 4 1.4 Histone Methylation ............................................................................................................. 5 1.5 SET Domain-containing Histone Methyltransferases and diseases ..................................... 8 1.6 Histone Lysine Methylation as a Signature for Gene Regulation ...................................... 10 CHAPTER 2 Function of SETD5 in mouse embryonic stem cell differentiation ....................... 13 2.1 Introduction ........................................................................................................................ 14 2.1.1 SET Domain-containing protein 5 (SETD5) ..................................................................... 14 2.1.2 Rationale and hypothesis ..................................................................................................... 17 2.2 Materials and Methods ....................................................................................................... 20 2.2.1 Antibodies, plasmids and other reagents ........................................................................... 20 2.2.2 Mouse ESC culture and AP staining .................................................................................. 21 2.2.3 Retinoic acid (RA)-induced differentiation of mouse ESCs ........................................... 21 2.2.4 RNA interference .................................................................................................................. 23 2.2.5 Western blot analysis ...........................................................................................................
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